A multi-stage anoxic/oxic (A/O) moving-bed biofilm reactor (MBBR) system with multiple chambers was established for municipal wastewater treatment. The active biomass quantity, bioactivity, and biomass yield of a pilot-scale multi-stage MBBR were investigated in this study. The microbial activity and heterotrophic yield coefficients (YH) were measured using respirometric techniques in each chamber at different temperature conditions. Meanwhile, the growth, nitrification, and denitrification rates of functional biomass were also quantified as specific respiration rates (SOURs). The total active biomass in the multi-stage A/O-MBBR system was 0.71–1.68 g COD/m2 for the aerobic reactor and 0.39–1.44 g COD/m2 for the anoxic reactor at 10–19 °C. The YH values for the anoxic reactors were 0.61–0.69, which were comparable to the recommended value of the activated sludge model (ASM1). The correlation coefficient between Nitrospira and the autotrophic specific respiration rate (SOURA) was 0.82. Meanwhile, denitrifying genera showed a significant correlation with the heterotrophic specific respiration rate (SOURH) and the active heterotrophic biomass (XH). This study provided insights into biomass distribution and the corresponding kinetic parameters for the multi-stage MBBR systems, which may serve as a reference for process design and troubleshooting.

  • The active biomass was quantitatively investigated in a multi-stage MBBR.

  • Microbial activity and heterotrophic yield coefficients were quantified in each reactor.

  • The use of respirometric techniques to assess microbial performance is feasible.

  • The correlation analysis of microbial community with active biomass and SOUR was carried out.

  • Data and facts were provided via long-term comprehensive investigation.

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